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Genotoxicity of Colloidal Fullerene C60 Shun Matsuda,† Saburo Matsui,‡ Yoshihisa Shimizu,† and Tomonari Matsuda*,† † ‡
Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 5200811, Japan Matsui Consulting Firm of the Environment, 10-45 Hanozonouchihata-cho, Ukyo-ku, Kyoto 6168045, Japan
bS Supporting Information ABSTRACT: Previous genotoxicity tests of aqueous fullerene C60 suspension (aqu-C60) yielded both positive and negative results. In the present study, aqu-C60 elicited positive responses in two bacterial genotoxicity tests, the Bacillus subtilis Rec-assay and the umu test at concentrations as low as 0.048 mg/L and 0.43 mg/L, respectively. In mammalian cell experiments, aquC60 showed a significant growth inhibitory effect on human hepatocarcinoma HepG2 cells at 0.46 mg/L. The level of the oxidative DNA lesion 8-oxo-7,8-dihydro-20 -deoxyguanosine, measured by liquid chromatography tandem mass spectrometry, was slightly but not significantly increased in HepG2 cells treated with 0.46 mg/L for 24 h, whereas the level of the lipid peroxidation-related DNA lesion R-methyl-γ-hydroxy-1, N2-propano-20 -deoxyguanosine was not changed. Under the same conditions, we did not detect any bulky DNA adducts, as measured by 32P-postlabeling/polyacrylamide gel electrophoresis analysis. Our data suggest that aqu-C60 has DNA-damaging potential and that the DNA damage is not due to covalent DNA adduct formation by C60 itself.
1. INTRODUCTION Fullerene C60 (C60) is one of the most attractive nanoparticles because of its unique physical and chemical properties and its burgeoning application to electronics, cosmetics, medicine, and so on. Greater use of C60 will lead to its increased emission into the environment and greater exposure opportunity for living organisms. Therefore, many researchers are concerned not only with the convenience of C60 but also its potential hazardous effects. Although C60 is insoluble in water, it can be stably dispersed in aqueous solution by simply stirring in water for a long time.13 This raises the possibility that C60 could remain a stable aquatic pollutant, prompting toxicological evaluation of aqueous C60 suspension (aqu-C60). Althougth there are several studies of the genotoxicity of aquC60, some test systems showed positive results1,4,5 but others negative.4,6,7 Therefore, further studies on the genotoxic potential of aqu-C60 are required. In the present study, we carried out an array of genotoxicity tests: 1) we used the Bacillus subtilis Rec-assay and the umu test to model induction of a DNA repair response caused by aqu-C60; and 2) we exposed mammalian cells to aqu-C60 to measure the levels of bulky DNA adducts by using 32P-postlabeling/polyacrylamide gel electrophoresis analysis as well as the lipid peroxidation (LPO)-related lesion R-methyl-γ-hydroxy-1,N2-propano-20 -deoxyguanosine (CdG) and the oxidative lesion 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) by using liquid chromatography tandem mass spectrometry (LC/MS/MS). r 2011 American Chemical Society
2. MATERIALS AND METHODS Materials. C60 was a kind gift from Dr. Hirohito Tsue (Kyoto University, Kyoto, Japan). Yeast extract and Bacto tryptone were purchased from BD Bioscience (Franklin Lakes, USA). S-9 mix was purchased from Wako. Alkaline phosphatase was purchased from SIGMA (St. Louis, USA). Micrococcal nuclease and spleen phosphodiesterase were purchased from Worthington (Lakewood, USA). Bacterial Strains. Salmonella typhimurium TA1535/pSK1002 strain for the umu test was a kind gift from Dr. Yoshimithu Oda (Osaka Prefectural Institute of Public Health, Osaka, Japan). For the Bacillus subtilis Rec-assay, the Bacillus subtilis H17 (Recþ) and M45 (Rec-) strains were used. Preparation of Aqueous C60 Suspension and Characterization. Before experiments, C60 was purified with HPLC. C60 was dissolved in toluene, and the solution was injected onto the Shimpack FC-ODS column (150 mm 4.6 mm) (Shimadzu, Kyoto, Japan) and subsequently eluted in an isocratic mode with 60% toluene in acetonitrile at flow rate of 1.0 mL/min with monitoring the absorbance at 333 nm. The peak showing UV spectrum at 333 nm of C60 was collected and evaporated to dryness. Received: November 1, 2010 Accepted: March 29, 2011 Revised: March 25, 2011 Published: April 11, 2011 4133
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Environmental Science & Technology Preparation of aqu-C60 followed Deguchi et al.8 with slight modification. Ten mg of C60 was dispersed in 100 mL of THF, degassed with a nitrogen purge for five hours, and stirred overnight in the dark. After filtration (pore size: 0.45 μm), an equal amount of water was added to the solution. The solution was evaporated to 90 mL by using a rotary evaporator, following which 20 mL of water was added. The evaporation and wateraddition steps were repeated twice. Finally the solution was evaporated to 100 mL and insoluble C60 in the solution was removed by filtration (pore size: 0.45 μm). UVvis spectra of the aqu-C60 were scanned within the wavelength of 220600 nm using Gene Spec V (Hitachi, Tokyo, Japan). Size distribution was determined by SALD-2100 laser diffraction particle size analyzer (Shimadzu, Kyoto, Japan). Concentration of the prepared aqu-C60 was determined by absorbance at 263 nm with molar absorbance coefficient reported by Mchedlov-Petrossyan et al. and Deguchi et al.8,9 (ε, 1.1 105 M1 cm1). Final concentration in toxicity tests was calculated from dilution magnification of the prepared aqu-C60. Bacillus subtilis Rec-Assay. Bacillus subtilis M45 (Rec-) strain is a recA gene deficient strain lacking DNA recombination repair system and SOS response induction. Consequently, Rec- strain is much more sensitive than Bacillus subtilis H17 (Recþ) strain to wide spectra of DNA damage such as DNA strand breaks, pyrimidine dimers, alkylations, cross-links, and bulky DNA adducts.10 Therefore, genotoxicity of the interested chemical can be judged to compare the survival curve of Rec- with that of Recþ. Both Recþ and Rec- strains were grown in LuriaBertani (LB) broth at 37 °C with vigorous shaking until the turbidity of the cultures at 595 nm reached from 0.1 to 1.0 using a microplate reader. The cultures were then diluted with LB broth until the turbidity at 595 nm ≈ 0.02. Four microliters of water (negative control), the aqu-C60 sample were pipetted into the wells of a 96well microplate. Fifty microliters of the diluted culture and 46 μL of LB broth were added to each well, and then the turbidity of the mixtures at 595 nm (A595 (before)) was measured. The microplate was sealed and incubated at 37 °C for 5 h with vigorous shaking. After incubation, the turbidity of the mixtures at 595 nm (A595 (after)) was measured. Survival of Recþ and Rec- strains was calculated using the following equation: Survival (%) = {A595 (after) - A595 (before)}sample/{A595 (after) - A595 (before)}control. Umu Test. Salmonella typhimurium TA1535/pSK1002 strain used for umu test is introduced a plasmid pSK1002 carrying a fused gene umuC-lacZ and the expression of umuC is inducible by these DNA-damaging agents. The strain enables us to judge genotoxicity of the interested chemical by measuring the β-galactosidase activity in the cells produced by the fusion gene. The detailed protocol of the umu test was described elsewhere.11 Four microliters of water (negative control) or the aqu-C60 sample and 96 μL of an exponentially growing culture of TA1535/pSK1002 for the S9-absent experiment or the mixture of the bacterial culture and the S9 mix at a ratio of 1.7:0.3 for the S9-present experiment were added to the wells of a 96-well microplate. 4-Nitroquinoline 1-oxide (4-NQO) and 2-aminoanthracene (2-AA) as positive controls for the S9-absent and the S9-present experiment, respectively, and dimethylsulfoxide (DMSO) as their negative control were used. After incubation, bacterial growth was measured as turbidity at 595 nm with a microplate reader. For chlorophenol red-β-D-galactopyranoside (CPRG), the absorbance at 540 nm was measured. The relative β-galactosidase activity (RGA) was calculated using the
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following equation: RGA (units) = A540 (CPRG)/A595 (growth turbidity). The values in this equation were corrected by subtracting the value of the absorbance blank. The experiment was performed independently three times. Cell Culture. Human hepatocarcinoma cell line HepG2 was obtained through the courtesy of the Cell Resource Center for Biomedical Research of Tohoku University, Sendai, Japan and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (IWAKI, Funabashi, Japan) or phenol red-free DMEM (for MTS assay) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, Carlsbad, USA) at 37 °C in a humidified 5% CO2 atmosphere. MTS Assay. HepG2 cells seeded in a 24-well plate were treated with 450 μL of culture medium plus 50 μL of aqu-C60 (final concentration, 0.46 mg/L) for 24 and 72 h. Cell viability after treatment was assessed by using an MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay according to the manufacturer’s instructions (Promega, WI, USA). Cell Treatment with aqu-C60 and DNA Extraction. HepG2 cells grown to 70% confluence in 100-mm dishes were incubated for 24 h with either 1 mL of water (negative control) or aqu-C60 (the final concentration, 0.46 mg/L) plus 9 mL of the culture medium. After the cells were harvested, DNA was extracted from the cellular pellet according to Ravanat et al.12 using NaI for DNA precipitation. The DNA pellet was dissolved in 200 μL of 0.1 mM desferrioxamine. The DNA concentration of the samples was calculated by measuring the absorbance at 260 nm. Enzymatic DNA Digestion. For LC/MS/MS experiments, each DNA sample (25 μg) was mixed with 15 μL of digestion buffer (17 mM sodium succinate, 8 mM CaCl2, pH 6.0) containing 22.5 units of micrococcal nuclease and 0.075 units of spleen phosphodiesterase. Together, three stable isotope-labeled DNA adduct internal standards, [15N5]-8-oxodG, and [15N5]-RS-methyl-γ-hydroxy-1,N2-propano-20 -deoxyguanosine (CdG1), and [15N5]-R-R-methyl-γ-hydroxy-1,N2-propano-20 -deoxyguanosine (CdG2), were also added to the solution. After incubation at 37 °C for 3 h, 1.5 units of alkaline phosphatase (SIGMA, St. Louis, USA), 5 μL of 20 mM ZnSO4, 10 μL of 0.5 M Tris-HCl, pH 8.5, and 67 μL of water were added. The mixture was then incubated for another 3 h at 37 °C. The digested DNA was concentrated to approximately 20 μL by speed-vac concentrator, and 100 μL of methanol was added to precipitate enzymes and excess salt. The supernatant was recovered, and the precipitate was washed by 100 μL of methanol. The supernatant and the methanol fraction were combined and evaporated to dryness. DNA Adducts Quantification. LC/MS/MS experiments were performed on a Shimadzu LC system (Shimadzu, Kyoto, Japan) and a Quattro Ultima Pt triple stage quadrupole mass spectrometer (Waters-Micromass, Milford, MA). The digested DNA samples were resuspended in 50 μL of 30% dimethyl sulfoxide (DMSO). 50 μL aliquots of sample were injected onto a Shim-pack FC-ODS column (150 mm 4.6 mm) (Shimadzu, Kyoto, Japan) and subsequently eluted in an isocratic mode with 2% methanol in water initially for 0.1 min, a linear gradient of 2% to 40% methanol in water from 2 to 40 min, and a subsequent linear gradient of 40% to 80% methanol in water from 40 to 45 min at flow rate of 0.4 mL/min. The mass spectrometric conditions were performed with the following parameters: ion mode, positive; capillary voltage, 3.5 kV; ion source temperature, 130 °C; desolvation gas flow rate, 700 L/h; cone gas flow rate, 35 L/h. The characteristic parameters for each DNA adduct 4134
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Figure 3. Aqu-C60 showed a positive genotoxic response in the umu test with S9- (a) or S9þ (b). 4- NQO and 2-AA were used as positive controls in the S9- and S9þ experiments, respectively. The values represent the mean of three independent experiments ( SD. Asterisks (*, **, and ***) denote p < 0.05, 0.01, and 0.001 calculated using Student’s t test versus the control.
Figure 1. Characterization of aqu-C60. (a) The color images of aquC60. (b) UVvis absorbance spectra of aqu-C60. (c) Particle size distribution of 2.3 mg/L aqu-C60, aqu-C60 in LB broth (2.3 mg/L) and aqu-C60 in DMEM (2.3 mg/L). The average particle size of C60 was determined to be 122 nm in water, 320 nm in LB broth, and 330 nm in DMEM.
Figure 4. Cytotoxicity of aqu-C60 against HepG2 cells. After cells were treated with 0.46 mg/L aqu-C60 or water (control) for 24 h (a) and 72 h (b), MTS assay was performed. The values represent the mean of three independent experiments ( SD. Asterisks (**) denote p < 0.01 calculated using Student’s t test versus the control.
authentic standards applied with isotope internal standards. DNA adduct levels in each sample were calculated as described in a previous report.13
3. RESULTS
Figure 2. Survival curves for Recþ and Rec- cells treated with aqu-C60 in the Bacillus subtilis Recassay. The values represent the mean of three independent experiments ( SD. Asterisks (*, **, and ***) denote p < 0.05, 0.01, and 0.001 calculated using Student’s t test between Recþ and Rec-.
measurement were as follows (cone voltage (V), collision energy (eV), base ion f product ion): [15N5]-8-oxodG (40, 12, 288.8 f 172.8), [15N5]-CdG1 and CdG2 (35, 10, 343.0 f 227.0), 8-oxo-dG (40, 12, 283.8 f 167.8), and CdG1 and CdG2 (35, 10, 338.0 f 222.0). The amount of each DNA adduct was quantified by calculating the peak area ratio of the target DNA adduct and its specific internal standard. Calibration curves were obtained by
Aqu-C60 Characterization. The aqu-C60 exhibited yellow color (Figure 1(a)). The UVvisible absorption spectra of the aqu-C60 shown in Figure 1(b) were consistent with the spectra shown in a previous report.8 Size distribution of the aqu-C60 ranged from 59 to 241 nm (Figure 1(c)). The average size was determined to be 117 nm. However, the distribution was shifted to larger (241554 nm) when aqu-C60 was dispersed in LB broth or DMEM. The average size was determined to be 320 nm in LB broth and 330 nm in DMEM. Bacterial Genotoxicity Test. The results of the Bacillus subtilis Rec-assay are shown in Figure 2. While aqu-C60 did not affect the survival of the Recþ strain even at 0.43 mg/L, the survival of the Rec- strain decreased in a concentration-dependent manner (Figure 2). The surviving fraction (67.7 ( 1.98%) of the Rec- strain at the highest concentration (0.43 mg/L) was significantly lower than that (96.7 ( 3.67%) of the Recþ strain (p < 0.001). The results of the umu test are shown in Figure 3. The RGA represents the relative genotoxic strength of the tested chemical. 4-NQO and 2-AA, which are positive controls for the S9-absent and S9-present experiments, respectively, produced dose-dependent increases in RGA. In the S9-absent experiment (Figure 3 (a)), the RGA of aqu-C60 was increased dose-dependently 4135
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Figure 5. DNA adduct levels of the oxidative lesion 8-oxodG (a) and the LPO-related lesions CdG1 and CdG2 (b). 70% confluent HepG2 cells in 100-mm dishes were incubated for 24 h with either 0.46 mg/L aqu-C60 or water (negative control). The cells were then harvested, and DNA adduct levels were measured. The values represent the mean of three independent experiments ( SD.
and showed a significant increase at the highest concentration tested (0.43 mg/L) (p < 0.01). However, the increase in RGA was not evident in the S9-present experiment (Figure 3 (b)). The results of the Bacillus subtilis Rec-assay and the umu test indicate that aqu-C60 elicits a genotoxic response in bacterial cells. Effect of aqu-C60 on Mammalian Cell Proliferation. Because it was reported that C60 tends to accumulate in the liver,14 we used human hepatocarcinoma HepG2 cells for a cell viability assay. The cells were treated with 0.46 mg/L C60 for 24 and 72 h, and then the absorbance at 490 nm was measured using MTS assay. While no effect was observed in cell viability after the short time aqu-C60 exposure (24 h), aqu-C60 produced a significant inhibitory effect on cell proliferation after long time aqu-C60 exposure (72 h) (Figure 4).
Table 1. Summary of Aqueous C60 Suspension Genotoxicity Testsa experimental system bacterial reverse
cell line or tissue
dose
Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA/pKM101
mutation assay
result
ref
5 mg/plate
7
1 mg/plate
6
Chromosomal Damage in Mammalian Cells human lympocytes
0.0022 mg/L
þ
1
FE1-Muta mouse lung
100 mg/L
þ
16
epithelial cells lung of male C57BL/6J mice
0.2 mg/mouse,
þ
4
micronuclei test
A549 cells
0.02 mg/L
þ
4
chromosomal
Chinese hamster CHI/IU cells
5000 mg/L
7
200 mg/L
6
14 μM
þ
5
lung (in vivo)
0.2 mg/mouse 4 times,
4
lung (in vivo)
intratracheal instillation 0.2 mg/mouse,
þ
4
16
Li þ, Lu þ, Co -
27
comet assay
intratracheal instillation
aberration test
Transgenic Mutagenesis Systems Gpt delta transgenic mouse Spi- mutation assay
primary embryo fibroblasts (in vitro)
Gpt mutation assay
intratracheal instillation FE1-MutaTM Mouse Cll mutation assay 8-oxodG level in DNA
lung epithelial cells (in vitro)
100 mg/L
liver (Li), lung (Lu), and colon
0.064 (Li), 0.64
(Co) of female Fisher 344 rats
(Lu, Co) mg/kg body weight, oral gavage Results from This Study
Bacillus subtilis Rec-assay
Bacillus subtilis H17 and M45
0.048 mg/L
þ
this study
Umu test
Salmonella typhimurium
0.43 mg/L
þ
this study
0.46 mg/L
this study
0.46 mg/L 0.46 mg/L
(
this study this study
TA1535/pSK1002 32
P-postlabeling
HepG2
Oxidative DNA adduct formation 8-oxodG CdG a
HepG2 HepG2
þ: positive/significantly increased: -: negative/not changed; (: tended to increase but the increase is not significant. 4136
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Environmental Science & Technology Quantification of Oxidative DNA Adducts and Bulky DNA Adducts. C60 is known as an ROS generator1517 and an
inducer of LPO.1823 ROS and LPO products can modify nucleic acid bases to form DNA adducts, such as 8-oxodG, implicated in genotoxicity.24 Therefore, we elucidated whether aqu-C60 can increase the levels of oxidative DNA adducts in human hepatocarcinoma HepG2 cells. As candidate oxidative DNA adducts we chose 8-oxodG, CdG1, and CdG2. The results of quantification of oxidative DNA adducts are shown in Figure 5 (a) and (b). The levels of CdG1 and CdG2 were essentially unchanged following aqu-C60 treatment as compared to the control; a slight but nonsignificant increase in the level of 8-oxodG lesions was observed. In the same condition, the cell viability of HepG2 was not changed (Figure 4 (a)). Bulky DNA adducts were also measured by using the 32P-postlabeling method, but we could not detect any bulky DNA adducts caused by aqu-C60 (Figure S1).
4. DISCUSSION The size distribution shift by LB broth and DMEM observed in Figure 1(c) is thought to be due to salt in the media as reported previously.25 The size of colloidal C60 particle in those test media (241554 nm) was considerably big. Although mammalian cells may take it by endocytosis, it is hard to imagine that the bacterial cells can intake such a big particle as it is. So that, we feel that only a small portion of the aqu-C60 was taken up by the bacterial test strains. The availability of the particle aqu-C60 by bacterial and mammalian cells needs to be elucidated in further study. In this study, we prepared aqu-C60 by the THFwater exchange method. It was reported that γ-butyrolactone (GBL), a toxic byproduct of THF, contaminated in the aqu-C60 was prepared by this method.26 We checked the contamination by using LC/MS/MS, and the concentration of GBL in our aqu-C60 solution was 0.064% (v/v). We also checked if this concentration of GBL affected the test results (Figures S2 and S3). The results indicated that the influence of the contaminated GBL could be negligible. A summary of the results of genotoxicity tests available in literature, together with our data, is presented in Table 1. Aqu-C60 elicited positive genotoxic responses in experimental systems which detect early events in mutagenesis, including DNA damage and DNA repair responses, such as the comet assay,1,4,16 the micronucleus test,4 the Bacillus subtilis Rec-assay, and the umu test. These lines of evidence support the hypothesis that aqu-C60 has DNA-damaging potential. On the other hand, the results from some other experimental systems have been negative or conflicting: bacterial reverse mutation assay (negative6,7), chromosomal aberration test (negative6,7), and transgenic mutagenesis systems (positive,4,5 negative4). As describe above, the mutagenic effect of aqu-C60 is not conclusive. That gives us an impression that the mutagenic effect of aqu-C60 might be modest. The result of 32Ppostlabeling/polyacrylamide gel electrophoresis analysis indicates that aqu-C60 does not make covalent DNA adducts. So that C60 seems to make DNA damage by some indirect mechanisms. ROS generation is considered an important property of C60. Two mechanisms of ROS induction by C60 have been reported. First, C60 is excited from the singlet state to the triplet state by light. The excited triplet state of C60 produces singlet oxygen by energy transfer.17 Second, the excited triplet state of C60 is converted to the reduced triplet state in the presence of reducing agents such as NADH. The reduced triplet state of C60 produces
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superoxide anion radical by electron transfer.15 It was reported that exposure to aqu-C60 increased LPO in human dermal fibroblasts, HepG2 cells, human astrocytes, the brain of juvenile largemouth bass, rat lung, and adult male fathead minnows, as measured by the thiobarbituric acid assay for malondialdehyde.2023 These ROS and LPO products can oxidatively damage DNA to form DNA adducts such as 8-oxodG. Folkmann et al. reported that the levels of 8-oxodG were increased in the liver and lung but not the colon of rats after intragastric administration of C60 suspended in both saline and corn oil.27 However, we did not observe significant changes in the levels of 8-oxodG in HepG2 cells after treatment with 0.46 mg/L aquC60 for 24 h despite the known ROS-generating ability of C60 (Figure 5). On the other hand, there are few studies on LPOrelated DNA adducts in connection with particle toxicology. The levels of LPO-related DNA adducts CdG1 and CdG2 were also not significantly changed in this study (Figure 5(b)). One of the possible reasons for this discrepancy is that refined systems for removing 8-oxodG28 and CdGs29 in mammalian cells could have overcome the increased levels of these DNA adducts induced by aqu-C60. Overall, our data suggest that aqu-C60 has DNA-damaging potential and that the DNA damage is not due to covalent DNA adduct formation by C60 itself. The mechanism by which aqu-C60 induces DNA damage and the resulting mutation needs to be elucidated in further study.
’ ASSOCIATED CONTENT
bS
Supporting Information. Material and methods and Figures S1-S3. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: þ81-77-527-6224. Fax: þ81-77-524-9869. E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by KAKENHI (18101003 and 18014009) and Grants-in-aid for cancer research from the Ministry of Health, Labor and Welfare, Japan. ’ REFERENCES (1) Dhawan, A.; Taurozzi, J. S.; Pandey, A. K.; Shan, W.; Miller, S. M.; Hashsham, S. A.; Tarabara, V. V. Stable colloidal dispersions of C60 fullerenes in water: evidence for genotoxicity. Environ. Sci. Technol. 2006, 40 (23), 7394–7401. (2) Brant, J. A.; Labille, J.; Bottero, J. Y.; Wiesner, M. R. Characterizing the impact of preparation method on fullerene cluster structure and chemistry. Langmuir 2006, 22 (8), 3878–3885. (3) Cheng, X. K.; Kan, A. T.; Tomson, M. B. Naphthalene adsorption and desorption from Aqueous C-60 fullerene. J. Chem. Eng. Data 2004, 49 (3), 675–683. (4) Totsuka, Y.; Higuchi, T.; Imai, T.; Nishikawa, A.; Nohmi, T.; Kato, T.; Masuda, S.; Kinae, N.; Hiyoshi, K.; Ogo, S.; Kawanishi, M.; Yagi, T.; Ichinose, T.; Fukumori, N.; Watanabe, M.; Sugimura, T.; Wakabayashi, K. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems. Part. Fibre Toxicol. 2009, 6, 23. 4137
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